The cytochromes P-450 are membrane bound hemoproteins abundant in the liver. These proteins play a critical role in the metabolism of many endogenous and exogenous compounds including steroid hormones and drugs. Reactions catalyzed by cytochromes P-450 often produce metabolites less bioactive and more readily eliminated than are the parent compounds. To date, there have been at least 21 cytochromes P-450 purified from rat liver, each with characteristic structure, substrate binding affinities, and in some cases, differential regulatory responses to drugs or other xenobiotics. Black and Coon (1986) Comparative Structures of P-450 Cytochromes. In Cytochromes P-450: Structure, Metabolism and Biochemistry. P. R. Ortiz Dey Montellano editor. Plenman Publishing Corp., New York. Because each cytochrome appears to reflect expression of a unique gene, a standard nomenclature has been proposed based on dividing the P-450 genes into families and subfamilies according to nucleotide sequence homology.
Research has shown significant interpatient differences in the liver concentrations and activities of some cytochromes P-450. For example, the deficiency in the ability to metabolize debrisoquine, an antihypertensive medication, has been shown to be inherited as an autosomal recessive trait and appears to result from one of several mutations in the gene coding for a single liver cytochrome P-450 (P-450 IID gene family). Mahgoub, A et al 1977. Polymorphic hydroxlation of debrisoquine. Lancet 2:584-586. This defective cytochrome has been related to adverse reactions to many medications as well as other medical risks.
Predictions of adverse reactions to medications will require safe and noninvasive means to phenotype individuals in vivo for each of the cytochromes P-450 that demonstrate interpatient differences in catalytic activity and which play important roles in metabolism of drugs. One approach illustrated by Hepner and Vesell, N-Engl J Med 291:1384-1388, is by the aminopyrine breath test in which carbon labeled N-dimethyl aminopyrine is administered to patients and, because the radio-labeled carbon atom in the cleaved methyl group is largely converted to bicarbonate in vivo, the rate of demethylation can be conveniently monitored as the rate of production of radio-labeled carbon dioxide in the breath. Unfortunately, aminopyrine is demethylated twice by at least two different forms of cytochrome P-450 which have not yet been identified.
It has recently been discovered that the elimination of cyclosporine A (CsA) from the human body depends on its metabolism in the liver by cytochrome P-450III. Kronbach T. et al, Cyclosporine metabolism in human liver: Identification of a cytochrome P-450III gene family as the major cyclosporine-metabolizing enzyme explains interactions of cyclosporine with other drugs. Clin Pharmacol Ther 1988; 43:630-5. Combalbert J. et al Metabolism of Cyclosporin A. IV. Purification and identification of the rifampicin-inducible human liver cytochrome P-450 (Cyclosporin A Oxidase) as a product of P450IIIA gene subfamily. Drug Metabolism and Disposition 1989; 17:197-207. P-450IIIA is a Phase 1 enzyme whose catalytic activity varies many fold among patients. Guengerich F. P. et al, Characterization of rat and human liver microsomal cytochrome P-450 forms involved in nifedipine oxidation, prototype for genetic polymorphism in oxidative drug metabolism. J Biol Chem 1986; 261:5051-60; Wrighton S. A. et al, Purification of a human liver cytochrome P-450 immunochemically related to several cytochromes P-450 purified from untreated rats, J Clin Invest 1987; 80:1017-22.
CsA is an immunosupressive drug widely used to prevent allograft rejection in transplant recipients and appears to be useful in the treatment of many common autoimmune diseases. Bach J-F. Cyclosporine in autoimmune diseases. Transplantation Proceedings 1989; XXI (Suppl 1):97-113. In an attempt to limit toxicity while maximizing the therapeutic effects of CsA, blood levels are usually closely monitored for at least several weeks after patients begin treatment with CsA, and the daily dose is adjusted to achieve a trough blood level within a relatively narrow range. Kennedy MS, et al, Correlation of serum cyclosporine concentration with renal dysfunction in marrow transplant recipients. Transplantation 1985; 40:249-53. Irschik E. et al, Cyclosporine blood levels do correlate with clinical complications. Lancet 1984; 2:692-3. Konigsrainer A. et al, Rigid-dose regimen versus blood level-adjusted cyclosporine in elderly cadaveric renal allograft recipients. Transplantation 1988; 46:631-44. Moyer T. P. et al, Cyclosporine nephorotoxity is minimized by adjusting dosage on the basis of drug concentration in blood. Mayo Clin Proc 1988; 63:241-7. This procedure is often a tedious and costly process because the daily dose of CsA required to achieve a target blood level can vary at least 10 fold among patients. Kahan B. D et al, Optimization of cyclosporine therapy in renal transplantation by a pharmacokinetic strategy. Transplantation 1988; 46:631-44. Thus, an empirical initial dose of CsA will produce either potentially sub-therapeutic or potentially toxic blood levels in many patients. Furthermore, because CsA has a relatively long blood half-life (Ptachcinski, R. J. et al, Clinical pharmacokinetics of cyclosporine. Clinical Pharmacokinetics 1986; 11:107-32), it can take many days or weeks to arrive at an appropriate CsA dosing regiment for some patients. At some medical centers, CsA pharmacokinetics are routinely determined to better estimate individual dosing requirements. These studies can take several days to perform and have not become a standard practice.
The U.S. Pat. No. 4,676,974 to Hofmann et al, issued June 30, 1987 discloses a breath test method for pancreatic exocrine function wherein a radioactive labeled ester such as cholesterol octanoate is ingested in the body and is hydrolyzed by pancreatic enzymes and oxidized to labeled carbon dioxide which is expired in the breath. The rate of the appearance of the labeled carbon dioxide in the breath is a function of the rate of hydrolysis of pancreatic enzymes which in turn reflects pancreatic exocrine function.
The U.S. Pat. No. 4,298,347 to Walsh, issued Nov. 3, 1981, discloses a method for the analysis of exhaled carbon dioxide for clinical diagnostic application which contains a mixture of .sup.13 CO.sub.2 and .sup.12 CO.sub.2. The method relates strictly to a carbon dioxide breath test but does not relate at all to the analysis of an enzyme function.
The U.S. Pat. Nos. 3,792,272 to Harte et al, issued Feb. 12, 1974 and 4,407,152 to Guth, issued Oct. 4, 1983 relate to breath test devices for detecting the concentration of ethyl alcohol in the breath.
Brueck et al, CHemical Abstracts 107, 53776s (1987) discloses a carbon-14 dioxide (.sup.14 CO.sub.2) breath test. The article only refers to the measurement of "modified" or "altered" P-450 activity. That is, the article addresses only changes in breath tests brought about by induction or inhibition of drug metabolizing enzymes, including P-450's, by the administration of other drugs. The article does not address the concept of quantitating the activity of a P-450 which uniquely catalyzes N-demethylation in "normal" individuals. The article does not discuss the idea that breath tests can specifically measure specific forms of P-450. The article merely speculates that the reason that phenobarbital treatment did not increase the production of CO.sub.2 from methoxy-acetanilid was due to alterations in the one carbon pool kinetics. However, another reason for this effect is that the form to P-450 that metabolizes methoxyacetanilid is not inducible by phenobarbital. Further, the article studies exclusively O-demethylation reactions and not N-demethylation. The only reference to humans in the article is a speculation that it may be possible to develop a diagnostic analysis for the metabolic disorders by means of a breath test technique but the article does not foresee or suggest that a breath test may be suitable for determining the activity of specific P-450's in healthy individuals.
Coates et al, Chemical Abstracts 96, 139-172h (1982) discusses the detection of deconjugation of bile salts with a carbon-14 labeled carbon dioxide breath test. The abstract does not relate at all or even suggest a test based on N-demethylation nor does it relate to or suggest a breath test that may be suitable for determining the activity of specific P-450's in healthy individuals.
The present invention relates to a novel approach for characterizing the major glucocorticoid-inducible cytochromes P-450 (P-450 IIIA a gene family) present in the livers and intestine of rats and man. Since P450IIIA enzymes catalyze and in most instances, limit the rate of erythromycin N-demethylation in liver microsomes prepared from their respective, the rate of production of labeled carbon dioxide in breath after injection of a test dose of radio-labeled N-methyl erythromycin provides a convenient and selective measure of the catalytic activity of these cytochromes in vivo.
Because P-450IIIA catalyses the N-demethylation of erythromycin, applicant reasoned that a patient's ability to demethylate erythromycin may be useful in predicting an appropriate dosing regiment for CsA.